Protozoa species were identified in rumen contents of four domestic sheep (Ovis aries) from South Africa. All animals were fed a forage diet which consisted of 50% lucerne and 50% teff hay. Ten new host records were identified, bringing the total number of species and forms observed in sheep in South Africa to 30. The occurrence and geographic distribution of ciliate protozoa in both domestic and wild sheep from around the world are summarised. It was found that 15 genera and 131 species occur in domestic sheep globally.

Introduction

Other than several early reports from South Africa on rumen ciliate protozoa in domestic sheep (Fantham 1920, 1921, 1926; Van der Wath & Myburgh 1941), no additional research in this respect has been published. These previous studies identified a total of only 12 species and forms. Studies from other areas of Africa are also limited (see, e.g. Selim et al. [1996] for Egypt and Selim et al. [1999] for Libya). To date only 17 species have been observed in domestic sheep across Africa.

Materials and methods

During August 2009 samples of rumen contents were obtained from four fistulated male domestic sheep (Ovis aries), which were all housed together in 6 m x 10 m pens at the Department of Anatomy and Physiology, University of Pretoria (Onderstepoort), South Africa. Half of each pen was covered by a concrete floor, whilst the other half was an open-air dirt floor. The animals were rotated to a clean pen every week. The sheep were fed a 50:50 mixture of lucerne (Medicago sativa) and teff hay (Eragrostis tef) each morning, after which they were allowed free access to kikuyu grass (Pennisetum clandestinum) pasture for the rest of the day. Rumen samples of 200 mL each were collected through rumen fistulas and 200 mL warm saline was added immediately to each sample. This helped to extend the cilia of the protozoa for easier identification. The sample was then preserved by adding 70% alcohol (100 mL).

Each sample was washed in a Pitchford-Visser filter, similar to the one used for trematode egg counts. The outer sieve had an aperture of 37 µm, whilst apertures in the inner sieve were 110 µm. This procedure retained all the coarse material in the inner sieve and the protozoa were captured in the outer sieve. The sample was recovered from the outer sieve by means of a drain tap. The washed sample was drained into a container, allowed to stand and settle for 15 min and then most of the supernatant was decanted. This allowed concentration of the sample to a final volume of 40 mL. A measured volume of alcohol was added to preserve the sample. A few drops of methyl blue stain were added to stain the protozoa and nucleus (Booyse, Boomker & Dehority 2010).

A 0.1-mL aliquot of stained sample was transferred onto a glass microscope slide using a 'Finnpipette', commonly used in chemistry laboratories. The sample was covered with a glass cover slip and examined with a standard Nikon microscope fitted with a Panasonic digital camera. Photographs were taken to aid identification. Each sample was examined in triplicate.

Owing to the observed lack of Entodinium spp. in the washed samples, additional samples of rumen contents from the four sheep were obtained. Sub-samples (0.1 mL) of these samples were stained and examined under the microscope to verify the presence of Entodinium spp., which had been lost through the 37-µm filter.

Ethical considerations

Housing and care of fistulated animals in the Department of Anatomy and Physiology were approved on an ongoing basis by the University of Pretoria Animal Care and Use Committee, as sheep are routinely used as rumen fluid donors, both for experimental and for teaching purposes.

Results and discussion

A total of 20 species and forms of protozoa were observed in the four sheep used in the present study. Of these species, 10 were identified in South Africa for the first time (Table 1). This brings the total number of species and forms reported from sheep in South Africa to 30. All Entodinium species listed in Table 1 were identified in whole rumen content (i.e. unfiltered samples). It is noteworthy that for these new host records in South Africa at least three of the Entodinium species are closely related or possibly similar to Entodinium dubardi (Dehority 1994). These are Entodinium caudatum f. dubardi, Entodinium parvum and Entodinium simplex. Entodinium nanellum would have been included in this group, but had been observed previously. Two of the other Entodinium species reported as a new host record, namely Entodinium exiguum and Entodinium longinucleatum, can occur on a variation line with E. dubardi. However, two rather distinctive features allow identification as a separate species: for E. exiguum, a straight oesophagus that does not curve towards the macronucleus, and for E. longinucleatum, a macronucleus extending the entire length of the cell.

The number of species and forms observed for other geographical locations range from fewer than 10 to 49, as shown in Table 2. The number found in the present study is about halfway between these extremes. In general, several reports where only a limited number of species were observed, appear not to be comprehensive studies; that is, only a few species were identified rather than a complete listing of all species present. Göçmen et al. (1999) observed that the protozoan fauna of sheep in Turkey and Far Eastern areas (e.g. China and Japan) appeared to be more diverse than in European and American sheep.

A number of the studies listed in Table 2 were reported between 1920 and 1985, before the most widely used classification scheme of Lee, Hutner and Bovee (1985) was published. Therefore, the older species names in the subfamily Diplodiniinae listed in the original reports have been corrected according to the more recent classification scheme. For example, Eodinium spp. are now classified under Diplodinium and Eremoplastron spp. under Eudiplodinium. Diploplastron affine is now classified as Metadinium affine. In more recent studies by Göçmen (1999a, 1999b), classification of both Epidinium spp. and Ophryoscolex spp. have been redefined and are used in Table 2. The total number of protozoa found in each of the localities indicated in Table 2 is summarised and listed at the end of the table.

Table 3 lists species and forms of protozoa observed in wild Bighorn and Dall sheep in the USA. Bighorn sheep are found in the Rocky Mountains from Colorado to Canada, whilst the Dall sheep live further north, reaching into Alaska. Many of the protozoa found in these two species (10 in Bighorn and 11 in Dall sheep) are also present in domestic sheep. Although not reported from domestic sheep, at least two species have been observed in Turkish cattle, namely Entodinium dalli, and a second form, Entodinium dalli rudidorosospinatum (Göçmen & Őktem 1996). Of particular interest is the presence of two new species of Polyplastron, neither of which has been observed elsewhere.

It is possible that as further in-depth studies are conducted, we will find a somewhat uniform distribution of protozoal species around the world.

Conclusion

We identified 10 species of rumen ciliate protozoa that had not been previously observed in rumen contents of South African sheep. This brings the total of species observed in this country to 30. The collection of protozoal fauna in South African sheep was compared to previous data reported for sheep worldwide and appeared to be somewhat less diverse.

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